Stability Of Horseradish Peroxidase Research Articles

Effect of PEGylation on the activity and stability of horseradish peroxidase and l-N-carbamoylase in aqueous phases

Effect of pegylation on the activities and stabilities of horseradish peroxidase (HRP) and n-carbamoyl-l-amino acid amidohydrolase (l-N-carbamoylase) was investigated. Pegylation was favored under alkaline conditions with high PEG/enzyme molar ratios. The lysine residues in l-N-carbamoylase were found to be more readily accessible for pegylation than those in HRP, leading to the formation of multi-pegylated enzymes. Pegylation and addition of mPEG as an additive improved HRP activity by 64% and 42%, respectively, via the formation of protective layers against the free radical-induced inactivation. The activity of pegylated l-N-carbamoylase, however, declined due to the additional mass transfer resistance introduced by the PEG layers. The extent of reduction in l-N-carbamoylase activity increased with the level of pegylation. In terms of thermostability, both the addition of mPEG as an additive and pegylation with PEG5000 led to a decline in thermostability. Nevertheless, pegylation of HRP with PEG2000 resulted in a PEG-HRP conjugate exhibiting a thermostability two-fold that of the control at 70°C, probably due to the capability of the relatively hydrophilic PEG domains on the conjugate in avoiding enzyme aggregation. After a 4-day incubation at room temperature, pegylated HRP retained significantly higher activity, 89%, than the control, 12%.

Horseradish Peroxidase‐Carrying Electrospun Nonwoven Fabrics for the Treatment of o‐Methoxyphenol

The carboxyl‐functionalized polystyrene (poly(styrene‐co‐methacrylic acid), PSMAA) nanofibers with average diameters of 250 ± 20 nm was prepared by electrospinning. PSMAA nanofibrous membrane were employed for immobilization of horseradish peroxidase (HRP) enzyme on the fibrous surface by a chemical method. The parameters about immobilizing HRP on the PSMAA nanofibers were studied and the influence on the activity of the HRP is discussed. This study showed that soap‐free emulsion method is an ideal technology to modify the polystyrene surface and ultimately achieve enzyme immobilization on electrospun PSMAA nanofibers surfaces. Compared with free HRP, the acid‐base stability, thermal stability, and storage stability of HRP were increased after the immobilization. The immobilized HRP maintained about 60% of its initial activity during a 20‐day storage period. However, the free HRP maintained only 40% of its initial activity. The removal percentages of o‐methoxyphenol (OMP) reached 80.2% after 120 min for immobilized HRP. These results suggest that the proposed scheme for immobilization of HRP has potential in industrial applications for the treatment of phenolic wastewater.

Efficiency of Carbohydrate Additives on the Stability of Horseradish Peroxidase (HRP): HRP‐Catalyzed Removal of Phenol and Malachite Green Decolorization from Wastewater

AbstractThe present study was undertaken to develop a cost effective stabilized horseradish peroxidase (HRP) for removal of phenol and decolorization of malachite green from synthetic wastewater. As revealed by enzyme stabilization assays, guar gum and galactose improved the stability of HRP successfully in solution state as compared as other additives such as pectin, carrageenan, or starch, respectively. In order to enhance the stability of HRP, combination of galactose and guar gum were effectively optimized together along with pH by constructing an appropriate response surface methodology (RSM). The RSM results suggest 18.25% of galactose and 0.35% of guar gum for maximum stabilization of HRP at 60°C, pH 7.4. In this study, optimization of removal of phenol and decolorization of malachite green using stabilized HRP was successfully carried out. Various process parameters such as aqueous phase of pH (7.4), concentration of enzyme (1.8 U/mL), phenol concentration (0.1 mM), and reaction time of 180 min were optimized effectively to achieve maximum removal of phenol from synthetic waste water. Dye decolorization studies indicated that stabilized HRP (1.2 U/mL) required lower concentration than the native enzyme (2 U/mL) to obtain similar decolorization efficiency. In addition, stabilized enzyme successfully decolorized the dye at 60°C, pH 7.2.

Enzyme–polysaccharide interaction: A method for improved stability of horseradish peroxidase

With the advent of green technology, use of enzymes as biocatalyst has become increasingly popular. However, in doing so, enzymes can lose their structure and catalytic activity under conditions that might be necessary for other components of processes. Compared to other strategies, chemical modification is a simple and effective technique for generating stable enzyme. Horseradish peroxidase (HRP; EC 1.11.1.7) was chemically modified by conjugating with 10 different polysaccharides. All polysaccharides were found to increase the thermal and pH stability of HRP with starch being most promising. Further, different parameters were evaluated for effective conjugation and thus stability of HRP conjugate. The degradation kinetics and storage stability of HRP proved the conjugate to be 6.4 times more stable than free enzyme. The starch conjugated HRP and free HRP were further evaluated for its application in decolorization of bromophenol blue dye. Both the enzymes were able to efficiently (>90%) decolorize the dye within minutes.

Modification of Lysine Residues of Horseradish Peroxidase and Its Effect on Stability and Structure of the Enzyme

Biotechnology is consistently seeking improved enzyme stability. Enzymes have great properties, although their marginal stability limits their applications. Among the strategies for improving stability of the enzymes, chemical modification is a simple and effective technique. In the present study, chemical modification of horseradish peroxidase (HRP) was carried out with 2,3-dichloromaleic anhydride and 2,3-dimethylmaleic anhydride. HRP is an important heme-containing enzyme. It is widely applied in pharmacological, chemical, and medical industries. Here, thermal stability of HRP was investigated at different temperatures. In addition, the enzyme stability was evaluated in urea, DMSO, alkaline pH, and hydrogen peroxide solutions by spectroscopic techniques. Structural investigation indicated that the both anhydrides slightly decrease compactness of the enzyme structure. The results also indicated that 2,3-dichloromaleic anhydride increases thermal stability of the enzyme and its stability in urea and DMSO solutions, but 2,3-dimethylmaleic anhydride only stabilizes HRP in urea solution. Furthermore, the experiments implied that none of the modifiers are effective on the stability of HRP in extreme pH and oxidative condition. Catalytic efficiency and activation energy did not change remarkably following reaction of the enzyme with the both carboxylic anhydrides. Consequently, improvement in the stability of HRP depends on not only the type of modifier but also denaturing condition.

Removal of phenols from acidic environment by horseradish peroxidase (HRP): Aqueous thermostabilization of HRP by polysaccharide additives

The bio-productive property of combinatorial polysaccharide additives (dextran and sodium alginate) on stability of horseradish peroxidase (HRP) for removal of phenols from acidic solutions was studied in this paper. The optimum pH range and temperature were determined for the stabilized enzyme as 3.6–5.4 and 65°C, respectively. Enzyme stabilization experiments were conducted in the solution state without enzyme immobilization or encapsulation. The combinatorial polysaccharides were selected to construct an appropriate response surface methodology (RSM) for maximum HRP stabilization together with sodium acetate buffer to optimize the polysaccharide additives. The RSM results suggest 10.08% of dextran, 0.41% of sodium alginate and 64mM sodium acetate buffer for maximum HRP stabilization at 65°C with a predicted percentage residual activity of 60.01%. DSC results corroborated that the denaturation temperature (TD) values of stabilized HRP to be 30°C higher than that of the native enzyme. The effect of pH on phenol removal for both native and stabilized HRP suggested that stabilized HRP exhibited high phenol removal activities even under acidic environment and successfully removed phenols.

A Bioelectrochemical Method for the Quantitative Description of the Hofmeister Effect of Ionic Liquids in Aqueous Solution

It is imperative to establish a simple, efficient, and practical method to investigate the Hofmeister effect of ionic liquids (ILs) on the behavior of proteins (enzymes). In this study, the effects of the cations and anions of different ILs in aqueous media on the structural stability of horseradish peroxidase (HRP), a model oxidoreductase, were systematically investigated using electrochemical methods. It is found that without ILs no direct electron transfer current signals of HRP appear at bare glassy carbon electrode (GCE) in phosphate buffer (pH 7.0) even after incubation and accumulation at a negative potential. In the presence of ILs, however, a current signal occurs at GCE, depending on the structure of the IL and its concentration. A linear relationship between the peak currents and the scan rates demonstrates that the direct electron transfer is a surface-confined thin-layer electrochemical process. The redox signal at GCE is from the heme of HRP. An IL has a perturbing effect on the HRP structure. The anodic peak current of HRP at GCE, the catalytic activity of HRP, and the secondary structure of HRP are well correlated. Different cations or anions at varied concentrations have different effects on the structural stability of HRP, resulting in different current signals at GCE. Thus, the anodic peak current of HRP at GCE can be used as an indicator to quantitatively characterize the effect of ILs on the structural stability of HRP. The present Hofmeister series for cations and anions is in good agreement with that reported elsewhere. To our knowledge, this is a first attempt to establish a simple and practical electrochemical method to correlate Hofmeister effects with characteristics of ions and solvents. The present investigation not only deepens our understanding of the complex electrochemical behavior of proteins in ILs media but also offers a practical guidance to designing "green" and biocompatible ILs for protein (enzyme) separation, purification, and enzymatic catalysis and conversion.

The Effect of Chemical Modification with Pyromellitic Anhydride on Structure, Function, and Thermal Stability of Horseradish Peroxidase

The stability of enzymes remains a critical issue in biotechnology. Compared with the strategies for obtaining stable enzymes, chemical modification is a simple and effective technique. In the present study, chemical modification of horseradish peroxidase (HRP) was carried out with pyromellitic anhydride. HRP has achieved a prominent position in the pharmaceutical, chemical, and biotechnological industries. In this study, the effect of chemical modification on thermal stability, structure, and function of the enzyme was studied by fluorescence, circular dichroism, and absorbance measurements. The results indicated a decrease in compactness of the structure and a considerable enhancement in thermal stability of HRP below 60 °C. It seems the charge replacement and introduction of the bulky group bring about the observed structural and the functional changes.

Effect of organic solvents on peroxidases from rice and horseradish: Prospects for enzyme based applications

A feasibility test for rice peroxidase (RP) enzyme as a substitute for horseradish peroxidase (HRP) was carried out. The activity of HRP was maximum at 30°C with pH 6.0–7.0. The purified rice peroxidase showed optimum activity at 30°C with pH 7–8 and was thermostable till 68°C, which is higher than the temperature reported for HRP. RP obeyed Michaelis–Menten kinetics. With increasing substrate concentrations, RP and HRP had Vmax as 8.23μMmin−1 and 4.21μMmin−1 and Km as 5.585 and 3.662mM, respectively. In 10% 1,4-dioxane and ethanol, RP exhibited 2 and 1.3 times higher activity, respectively than HRP. Shelf life studies show RP to be significantly stable till 60h in 20% 1,4-dioxane and till 12h in ethanol. The activity of RP/HRP increased gradually with 0%–40% ethanol or 0%–30% 1,4-dioxane till 20h with a sharp decline thereafter. The stability of HRP and RP reduced with increasing storage period. Enzyme efficiencies compared as Vm/Km showed water miscible organic solvents, viz.1,4-dioxane and ethanol, to exhibit a regular decrease in Vm/Km with increase in organic solvent concentration whereas, a reverse trend was observed with water–immiscible solvent like chloroform. The relative activity of RP and HRP enzymes upon immobilization on poly-5-carboxy-indole shows increasing enzyme activity with time and with guaiacol/dopamine hydrochloride as substrates. Immobilized RP had a better relative activity with dopamine as substrate than immobilized HRP, whereas with guaiacol both RP and HRP had a comparable activity upon immobilization. Results suggest rice peroxidase to be a cheaper and convenient enzyme system for immobilization using organic solvents. The high thermal stability, more stability in organic solvents and longer shelf life of RP over the immobilizing matrix suggest conducting polyindole having carboxyl functional groups to be a suitable matrix for the covalent entrapment of rice peroxidase through amide linkage. Good sensitivity and fast response to dopamine makes RP a suitable enzyme system for monitoring dopamine levels in aqueous medium.

Enhanced stability of horseradish peroxidase encapsulated in acetalated dextran microparticles stored outside cold chain conditions

Micro- and nanoparticles have been shown to improve the efficacy of safer protein-based (subunit) vaccines. Here, we evaluate a method of improving the vaccine stability outside cold chain conditions by encapsulation of a model enzyme, horseradish peroxidase (HRP), in an acid-sensitive, tunable biodegradable polymer, acetalated dextran (Ac-DEX). Vaccines that are stable outside the cold chain would be desirable for use in developing nations. Ac-DEX particles encapsulating HRP were prepared using two different methods, probe sonication and homogenization. These particles were stored under different storage conditions (−20°C, 4°C, 25°C or 45°C) for a period of 3 months. On different days, the particles were characterized for various physical and chemical measurements. At all conditions, Ac-DEX particles remained spherical in nature, as compared to PLGA particles that fused together starting at day 3 at 45°C. Furthermore, our results indicated that encapsulation of HRP in Ac-DEX reduces its storage temperature dependence and enhances its stability outside cold chain conditions. Homogenized particles performed better than probe sonicated particles and retained 70% of the enzyme's initial activity as compared to free HRP that retained only 40% of the initial activity after 3 months of storage at 25°C or 45°C. Additionally, HRP activity was more stable when encapsulated in Ac-DEX, and the variance in enzyme activity between the different storage temperatures was not observed for either particle preparation. This suggests that storage at a constant temperature is not required with vaccines encapsulated in Ac-DEX particles. Overall, our results suggest that an Ac-DEX based micro-/nanoparticles system has wide applications as vaccines and drug delivery carriers, including those in developing nations.

Nano reengineering of horseradish peroxidase with dendritic macromolecules for stability enhancement

A simple bio-conjugation procedure to surround a single horseradish peroxidase (HRP) enzyme molecule with dendritic polyester macromolecules (polyester-32-hydroxyl-1-carboxyl bis-MPA dendron, generation 5) was proposed. The characterization of resultant nanoparticles entitled HRP dendrozyme, was performed by transmission electron microscopy, dynamic light scattering, gel permeation chromatography and Fourier transform infrared spectroscopy. The results showed that HRP nanoparticles were spherical in shape and have an average size of 14 ± 2 nm in diameter. Furthermore, bio-conformational characterization of HRP dendrozyme was performed by means of circular dichroism and fluorescence spectroscopy to evaluate the secondary and tertiary structure changes after enzyme modification. These investigations revealed that protein conformation had small changes (in secondary and tertiary structures) after bio-conjugation. We also reported here that dendritic modification did not significantly affect the kinetic parameters of free HRP. The stabilization of HRP with dendron macromolecules as single enzyme nanoparticles resulted in improvement of half-life over 70 days storage at 4 °C as well as its tolerance under different elevated temperatures up to 80 °C and in the presence of organic solvents for 15 min. These significant results promise extensive applications of HRP particularly in harsh environmental conditions.

Poly(2-hydroxyethyl methacrylate) for Enzyme Immobilization: Impact on Activity and Stability of Horseradish Peroxidase

On the basis of their versatile structure and chemistry as well as tunable mechanical properties, polymer brushes are well-suited as supports for enzyme immobilization. However, a robust surface design is hindered by an inadequate understanding of the impact on activity from the coupling motif and enzyme distribution within the brush. Herein, horseradish peroxidase C (HRP C, 44 kDa), chosen as a model enzyme, was immobilized covalently through its lysine residues on a N-hydroxysuccinimidyl carbonate-activated poly(2-hydroxyethyl methacrylate) (PHEMA) brush grafted chemically onto a flat impenetrable surface. Up to a monolayer coverage of HRP C is achieved, where most of the HRP C resides at or near the brush-air interface. Molecular modeling shows that lysines 232 and 241 are the most probable binding sites, leading to an orientation of the immobilized HRP C that does not block the active pocket of the enzyme. Michaelis-Menten kinetics of the immobilized HRP C indicated little change in the K(m) (Michaelis constant) but a large decrease in the V(max) (maximum substrate conversion rate) and a correspondingly large decrease in the k(cat) (overall catalytic rate). This indicates a loss in the percentage of active enzymes. Given the relatively ideal geometry of the HRPC-PHEMA brush, the loss of activity is most likely due to structural changes in the enzyme arising from either secondary constraints imposed by the connectivity of the N-hydroxysuccinimidyl carbonate linking moiety or nonspecific interactions between HRP C and DSC-PHEMA. Therefore, a general enzyme-brush coupling motif must optimize reactive group density to balance binding with neutrality of surroundings.

Phosphate buffer effects on thermal stability and H2O2-resistance of horseradish peroxidase

Horseradish peroxidase (HRP) has attracted intense research interest due to its potential applications in biotechnological fields. However, inadequate stability under prevalent conditions such as elevated temperatures and H2O2 exposure, has limited its industrial application. In this study, stability of HRP was investigated in the presence of different buffer systems (potassium phosphate and Tris–HCl) and additives. It was shown that the concentration of phosphate buffer severely affects enzyme thermostability in a way that in diluted potassium phosphate buffer (10mM) half-life (from 13 to 35min at 80°C) and Tm (from 73 to 77.5°C) increased significantly. Among additives tested, trehalose had the most thermostabilizing effect. Exploring the role of glycosylation in stabilizing effect of phosphate buffer, non-glycosylated recombinant HRP was also examined for its thermal and H2O2 stability in both diluted and concentrated phosphate buffers. The recombinant enzyme was more thermally stable in diluted buffer in accordance to glycosylated HRP; but interestingly recombinant HRP showed higher H2O2 tolerance in concentrated buffer.

Additive Effect of Dextrans on the Stability of Horseradish Peroxidase

The influence of various concentration (10, 20, and 30% w/v) of different molar weighted dextrans as additives on the stability of HRP has been studied in aqueous medium. Native HRP preparations were formulated with different additives for storage stabilization and better performance at high temperature and pH. The results obtained show a stabilizing effect in the presence of an additive (75kDa dextran). The enzyme with 75kDa dextran (in concentration 10% w/v) showed the highest thermal resistance and the best performance for long-term storage at pH 5.0. In the presence of the 75kDa dextran, the enzyme activity was increased threefold at 25°C and lost only 15% activity in 2h at 50°C in comparison to the native enzyme which lost all its activity. In addition, dextran protected HRP against inactivation by air bubbles.

Stabilization of horseradish peroxidase in silk materials

Horseradish peroxidase (HRP) is widely used as an indicator enzyme in enzyme immunoassays, enzyme electrodes, effluent treatment and synthetic organics. Previous studies showed that HRP was not stable in solution especially for low concentration solutions. It is important to prevent HRP from losing its activity. In the present study, we describe HRP stabilization in silk fibroin solution and in silk films. The results showed that HRP activity increased 30%–40% after being added into silk solution. The half-life time of HRP activity was 25 days at room temperature and 1 h at 60°C in silk solution while it was only 2.5 h at room temperature and 0.3 h at 60°C in PBS buffer. The HRP activity in silk film still remained at 24%, 22%, 17%, when compared with the original amount of activity, after being immobilized in silk films stored at 4°C, room temperature and 37°C for 5 months, respectively. Electrostatic interactions and hydrophobic interactions between silk and HRP might account for this improved stabilization. Silk fibroin can be used as an HRP protecting reagent in solution and in films.

Stabilization of horseradish peroxidase by covalent conjugation with dextran aldehyde against temperature and pH changes

Abstract Stabilization of Horseradish Peroxidase (HRP; EC 1.11.1.7) against temperature and pH via the formation of the conjugates obtained by multipoint covalent bonding of dextran aldehyde (DA) to the enzyme were studied. Hence, three different molar weighted dextrans (17.5 kD, 75 kD, 188 kD) were covalently bonded to purified enzyme with different molar ratios (nHRP/nDA 20/1, 10/1, 1/1, 1/5, 1/10, 1/15, 1/20). The thermal stabilities of the obtained conjugates were evaluated with the activities determined at different temperatures (25, 30, 35, 40, 50, 60, 70, 80°C) applying 60 minutes incubation time. Conjugates formed were characterized by gel-permeation chromatography (GPC) and fluorescence techniques. The conjugate synthesized using dextran 75 kDa with nHRP/nDA 1/10 molar ratio showed better thermal stability than other conjugates and purified enzyme at pH 7. This conjugate also has wider activity pH range than purified enzyme. In addition, mentioned conjugate at pH 7 had very long storage lifetime compared to purified enzyme at +4°C and room temperature; which is considered a favorable feature for usage in practice.

Biocatalysis in Water-in-Ionic Liquid Microemulsions: A Case Study with Horseradish Peroxidase

In this article we report the first results on the enzymatic activity of horseradish peroxidase (HRP) microencapsulated in water-in-ionic liquid (w/IL) microemulsions using pyrogallol as the substrate. Toward this goal, the system used in this study was composed of anionic surfactant AOT (sodium bis(2-ethyl-1-hexyl)sulfosuccinate)/hydrophobic IL [C(8)mim][Tf(2)N] (1-octyl-3-methyl imidazolium bis(trifluoromethylsulfonyl)amide)/water/1-hexanol. In this system, the catalytic activity of HRP was measured as a function of substrate concentrations, W(0) (molar ratio of water to surfactant), pH, and 1-hexanol content. The curve of the activity-W(0) profile was found to be hyperbolic for the new microemulsion. The apparent Michaelis-Menten kinetic parameters (k(cat) and K(m)) were estimated and compared to those obtained from a conventional microemulsion. Apparently, it was found that HRP-catalyzed oxidation of pyrogallol by hydrogen peroxide in IL microemulsuions is much more effective than in a conventional AOT/water/isooctane microemulsion. The stability of HRP solubilized in the newly developed w/IL microemulsions was examined, and it was found that HRP retained almost 70% of its initial activity after incubation at 28 degrees C for 30 h.

Activity, stability, and unfolding of reconstituted horseradish peroxidase with modified heme

Heme-propionates of horseradish peroxidase (HRP) were esterified by p-nitrophenol, phenol and p-methylphenol to change its electron character and to increase its hydrophobicity. These synthetic hemes were inserted apo-HRP to give a novel HRP, respectively. Of the three reconstituted HRPs, reconstituted HRP with p-nitrophenol-modified heme derivative had a larger initial rate, affinity, catalytic efficiency and substrate-binding efficiency than native HRP in aqueous buffer and some solvents. The reconstituted HRPs showed higher thermostability and tolerance of DMF because of the increase of the hydrophobicity of the active site. Changing the electron character of the aromatic moieties linked at each terminal of the two heme-propionates can control activity and stability of HRP. The initial rate, affinity, catalytic efficiency and substrate-binding efficiency increased with the increases of electron-withdrawing efficiency of substituents at 4-position of the phenolic used to synthesize the heme derivatives, contrariwise, the stability decreased. The modifications resulted in the increase in the temperature ( T m) at the midpoint of thermal denaturation and the decreases in both enthalpy and entropy change at T m. The changes of catalytic properties and stabilities are related to the changes of the conformation of HRP. The modification changed the environment of heme and tryptophan, increased α-helix content of HRP. The present work demonstrates that enhancement of the hydrophobicity and the electron-withdrawing efficiency of heme improves the activity and stability of HRP.

Consensus mutagenesis reveals that non-helical regions influence thermal stability of horseradish peroxidase

The enzyme horseradish peroxidase has many uses in biotechnology but a stabilized derivative would have even wider applicability. To enhance thermal stability, we applied consensus mutagenesis (used successfully with other proteins) to recombinant horseradish peroxidase and generated five single-site mutants. Unexpectedly, these mutations had greater effects on steady-state kinetics than on thermal stability. Only two mutants (T102A, T110V) marginally exceeded the wild type's thermal stability (4% and 10% gain in half-life at 50 °C respectively); the others (Q106R, Q107D, I180F) were less stable than wild type. Stability of a five-fold combination mutant matched that of Q106R, the least-stable single mutant. These results were perplexing: the Class III plant peroxidases display wide differences in thermal stability, yet the consensus mutations failed to reflect these natural variations. We examined the sequence content of Class III peroxidases to determine if there are identifiable molecular reasons for the stability differences observed. Bioinformatic analysis validated our choice of sites and mutations and generated an archetypal peroxidase sequence for comparison with extant sequences. It seems that both genetic variation and differences in protein stability are confined to non-helical regions due to the presence of a highly conserved alpha-helical structural scaffold in these enzymes.

Effects of sodium phosphate buffer on horseradish peroxidase thermal stability

Thermal stability of horseradish peroxidase (HRP) was studied by differential scanning calorimetry, tryptophan fluorescence, the heme absorption and enzymatic activity analysis while the concentrations of sodium phosphate buffer ranged from 2.5 to 50 mM at pH 7.0. The results showed that the denaturation temperature (Tm) values decreased and the intrinsic tryptophan fluorescence intensity of denatured HRP increased as sodium phosphate buffer concentration increased. Furthermore, the heme absorbance at 403 nm and enzymatic activity of HRP decreased with the increasing buffer concentrations. According to data obtained in this experiment, it can be concluded that sodium phosphate accelerated the denaturation process of HRP and reduced the thermal stability of HRP.